幹細胞與生物材料於牙齒組織工程及再生

Abstract

牙齒常因牙周病、齲齒、外傷、腫瘤切除或遺傳疾病而喪失，若能發展出牙齒再生以取代喪失的牙齒是臨床上的新療法。本研究之目的是利用組織工程的方法達成牙齒的再生。牙髓幹細胞 (Dental pulp stem cell；DPSC) 為一具有多分化能力的幹細胞，在DPSCs分化成牙齒生成過程 (Odonteogenesis) 中，DPSCs可分泌許多生長因子 (Growth factor) 和形態原 (Morphogens)，這些生長及訊號因子與牙齒胚胎發育及生長所需是相同的，除細胞外，對牙齒組織工程來說，作為臨時細胞外基質的支架材料的選擇也是很重要的，一種理想的牙齒支架材料應具備無毒，無免疫致原與提供良好的生物相容性，利於細胞的貼附、增殖和分化，支架材料降解速率能與持續組織重建相互調控。故本實驗試著將DPSCs和滅菌牙本質(autoclaved treated dentin matrix；autoclaved TDM )支架;一起作用於牙齒再生研究。將牙齒組織切碎成＜1 mm3的小片段，於 DMEM培養液中加入 20％胎牛血清，以 37℃，5 ％CO2培養，待細胞量逹到1×106 cell/ml後，將 DPSCs全數打下並植入滅菌牙本質支架內，在以裸鼠小動物為寄主的研究模式中，將上述含DPSCs細胞之支架內以異種移植方式，移植到裸鼠後背部皮下處。並於 6週後犠牲，以 H&E和免疫組織化學染色評估牙齒再生部位。在以山羊為寄主的大動物研究模式中，以羊的 DPSCs植入人類的滅菌牙本質支架內先靜置1 天，再將 含DPSCs細胞之人類的滅菌牙本質支架亦以異種移植方式移植至羊隻齒槽骨內，3個月後以X光放射線攝影、H&E和免疫組織化學染色評估牙齒生長情形。在裸鼠結果顯示，在多數組別在 6週時可見到似牙本質-牙髓-結構，如牙本質小管( dentinal tubules)、前牙本質(pre-dentin)、似牙本質母細胞結構(odontoblast )、膠原蛋白纖維( collagen fibers)和血管可清楚辨識，且在免疫組織化學染色下表現出COLI、DMP、DSP、βⅢ tubulin，僅有未含細胞滅菌牙本質支架移植組別，結果並無顯現牙齒相關結構 。只有 DPSCs未移植支架顯現失序礦化基質之組織結構與纖維化組織。而在山羊的結果亦顯示，可在 X光放射線攝影下見到放射線不透明區域的出現。其中於 H&E切片染色下可觀察到牙髓腔、牙釉質、牙釉質母細胞、牙本質、牙本質母細胞、牙本質小管、牙骨質、血管和牙周韌帶的生成；而在免疫組織化學染色結果指出，牙本質、前牙本質、牙本質母細胞和牙本質小管皆表現 DSP、DMP；牙骨質可見到 CAP蛋白表現；牙釉質可見到 Amelogenien蛋白表現；牙周韌帶可見到 ScxA、βⅢ tubulin蛋白表現；牙髓腔內之血管內皮也表現出 factorⅧ蛋白。本實驗證實了利用DPSCs併用滅菌牙本質支架之組織工程方式，無論在裸鼠或山羊異體移植狀態下，皆可完成牙齒再生，亦進一步驗證了此 DPSCs併用滅菌牙本質支架組合可幫助牙齒相關組織的生成。

Periodontal disease, dental caries, trauma, tumor excision and generic disease may frequently result in tooth loss. Biological tooth substitute to replace lost teeth based on tissue engineering may provide a promising alternative strategy to currently clinical treatment. The purpose of this study is to develop a new tissue engineering method with the combination of dental pulp stem cells (DPSCs) and autoclaved treated dentin matrix scaffold (autoclaved TDM scaffold) in order to generate a complete tooth. DPSCs are multi-potential stem cells capable of differentiating into various cell types which exist in dental pulp. DPSCs can differentate to odontoblast when tooth growth during the odontogenesis. There are a lot of growth factors and morphogens in tooth development are match with odonteogenesis. Equally as important as the cells in the classic dental tissue engineering paradigm is the scaffold as a temporary extracellular matrix. An ideal scaffold should be non-toxic, non-immunogenic and biologically able to provide excellent cytocompatibility, and to support cell adhesion, proliferation and differentiation. The biodegradation rate of scaffold biomaterials should be controlled to allow a continuous tissue reconstruction. Autoclaved treated dentin matrix scaffolds based on extracted tooth from human or goat origin were retrieved, prepared and then minced dental pulp issues into < 1 mm3 pieces to culture in DMEM medium with 20％ FBS at 37℃ with 5 ％CO2 . When the DPSCs yield reached 1×106, the DPSCs were isolated, re-suspended, and then the cell suspension was injected into autoclaved treated dentin matrix scaffold. The DPSCs/autoclaved TDM scaffold constructs were implanted in xenograft into nude mice’s pelvic dorsum. After xenograft implantation for 6weeks, there were evaluated by hematoxylin and eosin stain and immunohistochemical (IHC). The results of nude mice showed 6 weeks expressed distinctive dentin-pulp-like structures, such as dentinal tubules, pre-dentin, polarizing odontoblast like structures, collagen fibers, and blood vessel were clearly observed. However, the control group (autoclaved TDM containing no cells) did not show the typical features of dentin and pulp tissues present. The control group comprising of DPSCSs with no scaffolds presented with disorderly mineralized matrix-like tissues with rich fibrous tissue. In the goat experimental groups, the radiographic analyses were performed, the results showed some opaque areas were formed. The generated pulp, enamel, ameloblast, dentin, odontoblast, dentin tubular, cementum, blood vessel, periodontal ligament and alveolar bone. The immunohistochemical analysis results showed the dentin, dentin tubular and odontoblast expressed DMP and DSP, periodontal ligament expressed ScxA, βⅢtubulin, cementum expressed CAP, and the vascular endothelial cell of pulp expressed factorⅧ, too. We demonstrated that dental pulp cells combined autoclaved TDM scaffold can regenerated tooth by tissue engineering when goat model with allogeneic cell transplantation and nude mice model with xenogeneic cell transplantation. We also demonstrated the dental pulp stem cells autoclaved TDM scaffold could promote the structures of tooth regeneration.